DE2438436B2 - Shaped article with an enzymatically active surface and process for its production - Google Patents

Description

The subject of the invention is "a molded object with an enzymatically active surface and a process for its production.

Enzymes fixed to a solid, insoluble carrier, so-called immobilized enzymes, acquire in many Application areas such as research, industry and medicine, increasing importance. The invnobilisicrtcn Enzymes become bound, entrapped, and crosslinked depending on the method of their immobilization Enzymes divided. Bound enzymes can be covalently bonded to active carriers, heteropolar Binding and / or van der Waals see interaction with ion exchangers and adsorbents obtained. as Trapped enzymes are found in crosslinked polymers, microcapsules, and regenerated cellulose derivatives mechanically immobilized enzymes called. Finally, enzymes can also be used with low molecular weight bisfunctional Reagenticn are networked and thus made insoluble.

A disadvantage of the immobilized enzymes is that the carriers suitable for fixation are often used mechanically unsatisfactory properties and not at all or only with difficulty Moldings with an enzymatically active surface can be processed. Because basically are only Such carriers based on plastic, biopolymers or inorganic substances are suitable for which reactive groups are present or can be generated and which groups or groups that are as hydrophilic as possible Centers included. On the other hand, for many applications, the immobilized enzymes are mechanical stable moldings required. Membranes, foils, tubes and other shaped bodies are suitable with an enzymatically active surface for many purposes the usual granulates and enable, for example, much higher flow rates of the substrate solution, reduced diffusion and use in fixed bed reactors. Also in the field of chemical analysis and therapy Such enzymatically active molded bodies would be very desirable in medicine.

It is already known tubular nylon hydrolyze on the surface and then fix it to the surface using glutaraldehyde enzymes.

ίο However, this procedure is relatively cumbersome and can not be transferred to other materials.

It is also known. Enzymes on the surface of molded articles, e.g. B. on glass spheres, through networking to bind with bisfunctional reagents such as glutaraldehyde. The disadvantage of this method is that the "enzyme film" is very sensitive and the enzymatic activity is a result of mechanical stress Denaturation of the enzyme is easily lost. In addition, this method is difficult to apply to carriers apply with hydrophobic surfaces.

From DE-OS 17 92 026 is also the chemical fixation of enzymes on sheet-shaped carriers, the surface of which has been substituted with reactive groups, such as triazinyl groups, are known. Here are the the same disadvantages as with the surface-linked enzymes. Also is the chemical fixation however, it can only be used with chemically appropriately substituted support material and the process can be used on supports with hydrophobic surfaces, even if they can be appropriately substituted, only with Apply difficulties.

It is known to coat a shaped body with a material suitable as an enzyme carrier and subsequent adsorptive or eovalen'.e binding of the enzyme to the carrier in the usual way.

All these processes have in common that the active soluble enzyme is close to the previously known methods of Enzyme immobilization is fixed on the shaped body. Since this fixation under precisely regulated Ambient conditions, especially with regard to pH and temperature, must take place. will the Production is very difficult and the yield is extremely low. These known methods hardly leave it either to fix several enzymes at the same time. Since an activated carrier must always be present here, comes further add that charged substrates or Kcakiantcn adsorbed, the pH optimum of the enzymes shifted and the Michaeliskonslanlen changed high loss of activity in the case of cross-linked enzymes accepted through irreversible engineering must be and the fixation is partly only adsorptive and therefore the activity is rapid bleeds out.

The object of the invention is therefore to eliminate these disadvantages and to provide a molded article with enzymatic to create an active surface that is as universal as possible in analytical and preparative chemistry as well as medical technology and which, on the other hand, can be manufactured in a simple manner can.

This object is achieved according to the invention by a molded article with an enzymatically active surface, which is characterized in that the surface of the opposite mold land is at least partially is covered with an adhesive layer and on the adhesive layer there are particles which at least contain an enzyme immobilized on a solid support.

The molded articles according to the invention can consist of any organic or inorganic material and can be shaped as desired. So The molded objects can be made of inorganic materials such as glass, metal, corundum or the like. Or made of an organic material such as natural and synthetic polymers, plastics and the like. Consist. They can be in the form of balls, tubes. Hoses, rods, foils, packing or any other forms exist for which an enzymatically active surface is desired, for example in Reaction tanks.

The surface of the molded article according to the invention is completely or partially covered by an adhesive layer overdrawn. Usually the adhesive layer will be present on those parts of the surface on which enzymatic activity is desired. The known and customary adhesives are used Types of glue in question. These include water-insoluble vegetable adhesives such as B. adhesives Rubber, natural resins or cellulose derivatives soluble in organic solvents, as well as synthetic ones Adhesives such as B. cellulose esters, butadiene-styrene and -Acrylnilril copolymers, polychlorobutadienes. Phenoplasts, e.g. B. Phenolformaldehyde Condensation Production and Rcsorcinfornialehyd condensation products. Aminoplasts such as I larmofl and M elaminfornialdchyd condensation products. Polyester, Polyisocyanates, epoxy resins, silicones, polyvinyl adhesives e.g. polyvinyl chloride, polyisobutylene, polystyrene, Polyvinyl alcohol, polyvinyl acetate. Polyvinyl ethers and polyacrylic and polymethacrylic acid esters as well their mixtures.

On the adhesive layer there are particles of enzymes immobilized on a solid carrier. In the context of the invention, these include customary enzymes immobilized on solid supports, in particular Understood enzymes covalently fixed to solid supports. For this, the well-known and partly in the trade to understand available immobilized enzymes, as for example in the German Oflenlcgungsschriftcn 21 28 743, 22 60 185, 19 08 290, 19 35 711 described are. In the context of the invention, preference is given to those particles which, according to the method of "Proieincopolymerisation" by introducing a polymerisable group into the enzyme and subsequent copolymerization with plastic-forming monomers can be obtained. Other types of immobilized enzymes can, however, also be used in the context of the invention.

The process for producing the molded articles according to the invention is that the liquid Adhesive applied to the surface of the molded item, allowed to dry, then particulate matter immobilized enzyme is applied and the adhesive is allowed to cure.

Surprisingly, the organic substances released during the setting and hardening of the adhesive Solvent or through the crosslinking reaction, the enzymatic activity of the applied immobilized enzyme is not destroyed, although the solvents commonly used in adhesives Denature enzymes and destroy their activity. So you have so far complicated and complex processes are used and the possibility of applying the enzymes simply by gluing passed by. The immobilized enzymes are expediently divided as finely as possible in the form Particles for the method of the invention inserted. Such fine-grained particles can easily pass through mechanical comminution of the usual immobilized enzyme parts, mostly in granulate form present, received. Preference is given to using immobilized enzymes that are finely divided into dust.

The application of the liquid adhesive to the surface of the molded article or the adhesive components takes place in the manner known and customary for this purpose. It is not necessary to go into more detail on this therefore. The drying is also done in the usual way until a possible high adhesiveness. The subsequent application of the particulate immobilized enzyme or the enzymes or enzyme mixtures can then be carried out by customary application methods, for example by inflating, sprinkling, immersing the molded article in the particulate immobilized Enzyme. Fluidized bed application and other comparable methods. For example be Hoses inside enzymatically active by blowing in powdery immobilized enzyme onto the with Adhesive coated inner surface, enzymatically active spheres. Rod. Packing. Stirrer and the like Applying or moving the shaped body in the particulate immobilized enzyme or a suspension received the same.

After the adhesive has cured, the moldings are preferably removed with a buffer solution of mechanically not lc-st bound particles and chemical components of the adhesive washed. Thereafter, the moldings obtained are usable immediately. But you can also under the usual conditions for carrier-bound enzymes be stored for a long time.

In the context of the invention it is also possible to use molded articles whose surface extends through a chemical or physical treatment can be converted into an adhesive in situ. This is for example with certain plastics such as polyvinyl chloride (PVC) possible by treatment with a suitable organic solvent and / or plasticizer or modifiers.

The particles of the carrier-bound immobilized enzyme used can be between about 0.0001 and about 1 mm, the range between 0.001 and 0.1 mm is preferred. The most suitable in each case Grain size of the particles depends on the fixed enzyme, from the respective carrier as well as from the adhesive, in particular from the organic one used in the adhesive Solvent. In the case of particularly sensitive enzymes, a particle size in the upper half of the specified grain size range preferred. | c the more stable the immobilized enzyme, the lower it can be also be the grain size.

It is a major advantage of the invention. that enzymatically active moldings are created that are practically independent of the physical properties in terms of their physical properties Properties of the "primary carriers", i. H. the for the Immobilization of the enzyme used particulate carrier material. The actual film body does not need to be converted into an active form and basically all immobilization methods Carrier-bound enzymes produced for the enzymatic activation of the surface of the molded body insert. Except for covalently bound enzymes, preferably covalently by protein copolymerization

bound enzymes can also be immobilized by the inclusion of enzymes within the scope of the invention can be used advantageously.

As already mentioned, the moldings according to the invention are suitable for a large number of types of application. For example, they can be in the form of hoses. Membranes and other shaped bodies in devices for enzymatic analysis can be used. For example, it is possible to use Tubing material with enzymatically active inner surface using enzymatic analyzes carry out conventional photometer, which work with flow cuvettes. Another possible application is in automatic analyzers with Dialysis membranes work, the use of enzymatically active dialysis membranes. For simple Detection reactions, enzymatically active test tubes and microscope slides can be used. For Moldings according to the invention are particularly suitable for use in preparative chemistry as reaction vessels, Pipelines and column packing cr.

The following examples further illustrate the invention.

example 1

Enzygel Glucosc-Oxidase / Catalase on tubes
Source material

Hose on polyvinyl acetate-polyethylene copolymer, internal diameter 1.5 mm

5 ml synthetic rubber-based adhesive
(Uhu-Kontakt® 2000)

10 ml of methylene chloride

100 mg of a glucose oxide as covalently bound on a cross-linked PolyacrylaiV.iJ carrier
(GOD) catalase (Kat) mixed enzyme according to DE-OS
(Enzygel® GOD / Kat), finely powdered

method

The glue solution diluted with methylene chloride is suspended vertically with a syringe Filled with hose (length 70-70 cm). As soon as the solution has run through completely (after 1–2 min) a conical pipette tip open on both sides with its lower opening into the tube introduced. The plastic pipette cone contains the enzyme powder. To the other part of the cone with a A pressure hose is connected with an opening of approx. 8-12 mm. Then the enzyme powder is used Approx. 1 -3 atü compressed air or nitrogen blown into the adhesive-coated hose. Excess enzyme powder can be caught at the bottom of the hose and used again.

After drying (approx. 1 hour), the tube covered with enzyme powder is rinsed with buffer solution until no more enzyme particles detach. The finished enzyme tube is then used for the automatic analysis of glucose. A piece of tube approximately 48 cm long shows a sample frequency of 24 / hour. and a ratio of sample to wash of 1: 4 as shown in FIG. 1 activity shown in the drawing. The sample frequency can be set to approx. 40 / hour. increase.

The analysis was carried out with a device arrangement according to the scheme shown in Figure 2 of the drawing.

The details of the device and measurement arrangement are given below:

Photometer: Lppendorf 1101 M
Flow cell: Hcllma No. 178,

1 cm layer delivery
Pump: Ismatic MP 13G) -IO
Coneclor (air supply): DO, H,

Tcchnicon no. llb-0203-00
Coil: 14 turns.Tcchnicon,

inner diameter 2 mm
Air separator: C-5,

Technicon No. 116-0202-05
Hoses:!. Air, inner diameter I mm

2. Sample, inner diameter 2.5 mm

3. Reagent, inner diameter 2.5 mm

4. Enzyme tube, internal diameter 1.5 mm. Length 48 cm

Sample solution: glucose, 0.25 mg / 100 ml -

200mg / 100mlinO, 1 M

Phosphate buffer. pH = 7.0
Reagent: 1.1 g of 2,2'-azino-di- [3-ethyl-bcnz-

thiazoline] sulfonate (6) (ABTS) + 0.6ml

Peroxidase (Boehringer, POD-I) in 1000 ml

0.1 M phosphate buffer
Measuring radiation: 405 nm. Temperature 25 "C

As with the Technicon-Auto-Analyzer®, both the sample and the reagent solution are in the tube segmented by air bubbles and continuously the substrate concentration by comparison with a standard solution measured.

Like F i g. 2 of the drawing shows, the sample solution (2) is filled with air bubbles before it enters the single-yeast reactor segmented (1) and then in the enzyme tube (or from a spiral milled in a bifilar present enzyme film) implemented. By adding reagent solution (3), the color reaction (or other Indicator reactions), which after the exit of the air bubbles (immediately before the Durchflußküvcltc) photometrically can be measured.

The deflections recorded with a recorder are directly proportional to the substrate concentration.

Example 2
Glucose oxidase / catalase on foil

Source material
Polyamide-polyester random fiber fleece

(Viledon® nonwoven, H 3003)
5 ml glue as in example 1
7 ml of methylene chloride

500 mg of the same enzyme particle preparation
as in example 1

method

The adhesive is diluted with methylene chloride and spread thinly on the film . After some time, the finely divided enzyme powder is applied to the still moist and sticky film using a sieve. After slowly drying, the film is slowly stirred in a 0.1 M phosphate buffer solution, pH = 7.0, and the unbound particles are removed in this way. This film is then milled into a bifilar

Spiral transferred and used in a similar way to the enzyme semicircle for determination of the substance.

1 cm ^{2 of} the film produced by the above-mentioned process weighs 23.3 mg in the dried state and shows an enzymatic activity of 16.1 U / g. corresponding to 0.188 U / cm ² .

Example 3
Coating a glass tip

Starting maicrial
I ml of glue as in Example 1
1.5 ml methyl chloride
50 mg enzyme powder as in example 1

method

The glass tip is immersed 1 to 2 times in the diluted adhesive solution and after a few seconds dipped in the enzyme powder. After slow drying, it is dissolved in a 0.1 M buffer solution. pH = 7.0 transferred and tested after approx. 12 hours. With a tip 1–2 mm in diameter and a length of 2-3 mm an average activity of 0.5 U / peak is obtained.

Example 4

A round polystyrene rod 2.4 mm in diameter was coated with an adhesive obtained from 6 g of a PVC adhesive (Tangit from Henkel) and 2 ml of tetrahydrofuran. The rod was then immersed in a finely powdered enzyme preparation which had or consisted of GOD (230 U / g) and Kai (3600 U / g) fixed on a polyacrylamide carrier by means of acrylosychloriJ according to the process of 'InzyrncopG-polymerization. It was then allowed to dry, washed with buffer and the activity determined. For GOD it was 0.49 U / ml ² surface area.

Example 5

The procedure of Example 4 was repeated using an alkaline phosphatase bound on the same support and having an activity of 200 U / g. The polystyrene rod was allowed to air dry for 15-30 seconds after immersion in the adhesive, then the enzyme dust was sprinkled on, allowed to dry and washed with buffer as noted above. The activity found was 0.23 U / mm ² surface.

Example 6

The process of Example 5 was repeated using an enzyme powder which contained 5 U / g trypsin on a maleic anhydride-acrylamide carrier according to DE-AS 19 08 290. The activity of the shaped body was 0.003 U / mm ² surface.

Example 7

Example 5 was repeated using a two-component epoxy resin adhesive (Uhuplus®) and the GOD / Kat powder from Example 4. The measured activity of the molded body was 0.5 U / mm ² surface.

Example 8

The process of Example 5 was repeated using a contact adhesive based on polychlorobutadiene with the addition of resins and organic solvents (Pattex * der l-'a. Henkel) and the enzyme powder from Example 4. The measured activity of the molding was 0. 45 rev / mm ² .

B c i s ρ i e I 9

The procedure of Example 8 was repeated using the enzyme powder containing alkaline phosphatase as the enzyme according to Example 5. The measured activity of the molded body was 0.021 U / mm ² .

'"Example 10

Example 5 was repeated using a commercially available adhesive based on a polyvinyl resin with acetone and ethyl acetate as the solvent (Uhu all-purpose adhesive) and the enzyme powder from Example 4. The enzyme activity measured on the molding was 0.5 U / mm ² .

Example 11

Example 10 was repeated using the enzyme powder from Example 6. The activity measured was 0.0026 U / mm ² .

Example 12

A PVC round rod with a diameter of 3 mm was partially dissolved on the surface with cyclohexane until the surface was sticky. The enzyme powder from Example 4 was then sprinkled on, the rod allowed to dry and washed with buffer solution. The enzymatic activity of the molding was 0.38 U / mm ² .

Example 13

Example 12 was repeated using the enzyme powder with alkaline phosphaase according to Example 5. The enzymatic activity of the shaped body was 0.117 U / mm ² .

Example 14

The process of Example 12 was repeated using the enzyme powder containing t-ypsin from Example 6. The enzymatic activity of the shaped body was 0.0042 U / mm ² .

Example I 15

A glass rod 2.4 mm in diameter was dipped into the adhesive from Example 8, then left to air dry for 15 to 30 seconds and the enzyme powder from Example 4 was sprinkled with it. After drying the rod, it was washed with buffer. The enzymatic activity of the finished molding was 0.66 U / mm ² .

B is ρ ie 1 1 6

A steel wire 1 mm in diameter was dipped into the adhesive from Example 8, allowed to air dry for 15 to 30 seconds, and then sprinkled with the enzyme powder from Example 4. The enzymatic activity of the finished molding was determined to be 0.80 U / mm ² .

Example 17

The procedure of Example 16 was repeated

using the enzyme powder with alkaline phosphatase according to Example 5. The enzymatic activity of the shaped body was determined to be 0.003 U / mm ² .

ίο

IJ e i s ρ i e I 18

Example 4 was repeated, but instead of a 1'olystyrene rod, a Teflon-coated magnetic stirring rod of 6.5 × 10.9 mm was used. The cn / ymatic activity of the molding was determined to be 0.15 U / mm ² .

Example 19

Example 5 was repeated, but the Teflon-coated magnetic stirring bar from Example 18 was used instead of the polystyrene rod. The enzymatic activity of the molded article was 0.135 U / mm- '.

For this purpose 2 sheets of drawings

Claims (5)

Patent claims: 1. Shaped article with an enzymatically active surface, characterized in that the surface of the molded article is at least partially coated with adhesive and on the Adhesive layer there are particles which are at least one immobilized on a solid support Contain enzyme. 2. Shaped article according to claim 1, characterized in that the base material made of plastic, Is made of metal or glass, the adhesive consists of a synthetic adhesive and the carrier of the immobilized enzyme consists predominantly of acrylamide units. 3. Process for the production of the molded article according to claim 1 or 2, characterized in that the liquid adhesive is applied to the surface of the Bred the opposite side of the mold, allowed to dry, then applied particulate immobilized enzyme and the adhesive is allowed to cure or dry. 4. Use of a molded article according to claim 1 or 2 as a reactor in the chemical or clinical enzymatic analysis. 5. Use of a molded article according to claim 1 or 2 as an enzyme reactor in the preparative chemistry.